1. Field of the Invention
The invention relates generally to mesenchymal cells and endothelial progenitor cells, and more specifically to methods for the isolation of mesenchymal cells and vascular progenitor cells from umbilical cord tissue.
2. Background Information
It has already been established that an excellent source for Mesenchymal Stromal or progenitor or stem cells is the Wharton's jelly which is the stroma of the umbilical cord. The umbilical cord originates from the extraembryonic mesoderm at day 13 of embryonic development, and is composed of two arteries and one vein, all of which are surrounded by the Wharton's Jelly which is a unique connective tissue stroma rich in proteoglycans and mucopolysaccharides. These stromal cells embedded in the collagen-rich matrix are myofibroblasts rather than typical fibroblasts. In recent studies, cord stromal cells were reported to possess mesenchymal stem cell character with, the differentiation of umbilical cord stromal cells into mesenchymal cell lineages, such as cardiomyogenic, chondrogenic, osteogenic, and adipogenic types. The isolation, culturing, and differentiation behavior of human perivascular umbilical cells and obtained osteogenic nodules has already been demonstrated by several researchers. The differentiation capacity of human umbilical cord mesenchymal stem cells into dopaminergic neurons has also been demonstrated. Most recently, some of embryonic stem cell markers, such as Oct-4, Sox-2, and Nanog, were demonstrated in porcine umbilical cord matrix cells. Similarly, endothelial progenitor cells obtained from the vascular system of the umbilical cord have been shown to possess the properties of similar progenitor cells obtained from other sources.
Mesenchymal stromal cells (MSCs), as defined by the International Society for Cellular Therapy, are plastic-adherent cells with a specific surface phenotype and have the capacity to proliferate (self-renew) and to differentiate into various lineages including bone, cartilage, adipose. Such cells can be derived from several different sources, such as trabecular bone, adipose tissue, synovium, skeletal muscle, dermis, pericytes, blood, and bone marrow. MSCs derived from bone marrow and adipose tissue have been studied extensively. MSCs derived from bone marrow can be differentiated into bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic cell-supporting stroma. Thus, they are candidates to treat patients suffering from bone disorders, heart failure, etc. Because MSCs can be isolated from adults in significant number, they have been examined closely for therapeutic utility. For example, MSCs support the ex vivo expansion of hematopoietic stem cells, act as immune modulators, release cytokines and growth factors, and they home to sites of pathology. A significant number of trials are ongoing using bone marrow-derived MSCs for a variety of indications, for example, acute myocardial infarction, stroke, and graft versus host disease. Nevertheless, there are limitations associated with MSCs derived from bone marrow for cell-based therapy. For example, collection of MSCs from bone marrow is an invasive and painful procedure. In normal aging, the marrow cavity fills with yellow fat. Thus, there may be difficulty in obtaining MSCs from older individuals. Along these lines, differences have been found between bone marrow-derived MSCs collected from a fetus versus adult-derived MSCs. For example, fetal MSCs have a longer life in vitro compared to adult-derived MSCs and MSCs derived from adults have a useful lifespan in vitro of about five passages.
Researchers have demonstrated that cells derived from the Wharton's Jelly in umbilical cords (so called umbilical cord matrix cells or UCMSCs) have properties of MSCs. While UCMSCs have surface phenotype, differentiation capability, and immune properties similar to MSCs derived from bone marrow and adipose, UCMSCs are more similar to fetal MSCs in terms of their in vitro expansion potential. In contrast to bone marrow- and adipose-derived MSCs, UCMSCs are isolated from the umbilical cord following birth and may be collected following either normal vaginal delivery or caesarean section. UCMSCs are easily expandable in vitro, and may be cryogenically stored, thawed, and reanimated. UCMSCs grow as plastic-adherent cells, express a surface phenotype similar to other MSCs, and differentiate into multiple lineages. Umbilical cord matrix cells have been safely transplanted and ameliorated symptoms in an animal model of Parkinson's disease, neural damage associated with cardiac arrest/resuscitation, retinal disease, and cerebral global ischemia.
Umbilical Cord MSCs. MSCs have been isolated from several compartments of the umbilical cord (FIG. 1). Specifically, the MSCs have been isolated from umbilical cord blood, umbilical vein subendothelium, and the Wharton's jelly (FIG. 1). Within Wharton's jelly, MSCs have been isolated from three relatively indistinct regions: the perivascular zone, the intervascular zone, and the subamnion. It is unknown whether MSCs isolated from the different compartments of the umbilical cord represent different populations. This discussion is confined to the MSCs isolated from Wharton's jelly cells (WJCs; zones 3-5 in FIG. 1). A sub-population of the Wharton's Jelly cells (WJC) display MSC surface markers, suggesting that they are of the MSC family.
WJCs have stromal support properties deriving from the early stages of embryogenesis as extraembryonic mesenchyme, that is, primitive Wharton's jelly, surrounding the migrating embryonic blood island cells during their migration to the aorta-gonad mesonephros (AGM) from the yolk sac region. WJCs retain this property as demonstrated by their role in ex vivo hematopoietic expansion and in vivo engraftment of HSCs. It has been reported that WJCs produced cytokines similar to those of BMSCs and that WJCs synthesized granulocyte macrophage colony stimulating factors (GM-CSF) and granulocyte colony stimulating factors (G-CSF) that BMSCs did not. WJCs differ from BMSCs because WJCs are slower to differentiate to adipocytes. Since this and other features (listed below) are shared with MSCs derived from umbilical cord blood (UCB), it is unclear whether the MSCs derived from UCB differ from those found in Wharton's jelly. UCB-MSCs and WJCs have several common properties, such as poor ability to differentiate to adipocytes, shorter doubling times than BMSCs, and greater numbers of passages to senescence. Like WJCs, UCB-MSCs may make GM-CSF, although this has not been consistently found. It is clear that HSCs can be expanded by MSCs from both UCB and WJC (FIG. 1).
Compartments within the umbilical cord. Five separate regions have been shown to contain mesenchymal stromal cells: (1) MSCs can be isolated from 20-50% of freshly prepared mononuclear cell fractions from umbilical cord blood; (2) MSCs have been isolated from umbilical vein subendothelial layer; (3) MSCs can be isolated following enzymatic digestion of the outer layers of umbilical vessels, for example, the perivascular region; (4) intravascular space consistently produces MSCs in healthy individuals; (5) the subamnion region. Wharton's jelly includes zones 3 through 5. This review focuses on Wharton's jelly-derived cells and not on MSCs derived from umbilical cord blood (zone 1) or umbilical vein subendothelium (zone 2).
There are differences between BMSCs, UCB-MSCs and WJCs. First, the isolation frequency of colony forming units (CFU)-F from bone marrow is estimated to be in the range of 1-10 CFU-F per 106 mononuclear cells (MNCs) and in umbilical cord blood is reported to be around one CFU-F clone per 108 MNCs to 1-3 CFU-F per 106 MNCs. [Note: The isolation frequency from first semester foetal blood-derived MSCs was 8.2 CFU-F per 106 MNCs]. In contrast, cells derived from Wharton's jelly have a higher frequency of CFU-F. Thus, an order of magnitude more of MSCs than of Wharton's Jelly-Derived Stromal Cells bone marrow or umbilical cord blood may be found in the initial isolation from WJ. Second, coupled with the greater CFU-F frequency, the doubling time of WJCs and UCB-MSCs is shorter than adult bone marrow-derived MSC (BMSCs). Faster doubling time is a common feature for MSCs derived from foetal blood, cord blood, and Wharton's jelly, and this common feature is thought to reflect the relatively primitive nature of these MSCs compared to adult BMSCs. An important difference between UCB-MSCs and WJCs is that WJCs can be isolated from close to 100% of the samples, even from umbilical cords that are delayed in their processing up to 48-hour.
Wharton's jelly cells, like bone marrow stromal cells and other mesenchymal cells, are plastic adherent, stained positively for markers of the mesenchymal cells such as CD10, CD13, CD29, CD44, CD90, and CD105 and negatively for markers of the hematopoietic lineage. Moreover, WJCs morphologically resemble MSCs and can be expanded more than bone-marrow derived MSCs in culture. Human WJCs express precursor cell markers such as nestin. WJCs can be induced to form adipose tissue, bone, cartilage, skeletal muscle cells, cardiomyocyte-like cells, and neural cells and are amenable to biomedical engineering applications. Therefore, these cells fit into the category of primitive stromal cells; and, because Wharton's jelly is a plentiful and inexpensive source of cells, it appears to potentially impact fields such as regenerative medicine, biotechnology, and agriculture. Further work is needed to determine whether WJCs engraft long-term and display self-renewal and multipotency in vivo and, as such, demonstrate that WJCs are a true stem cell population.